This invention relates to monoclonal antibodies (mAbs) that bind to a human coagulation factor or cofactor and their use as self-limiting inhibitors of thrombosis.
Under normal circumstances, an injury, be it minor or major, to vascular endothelial cells lining a blood vessel triggers a hemostatic response through a sequence of events commonly referred to as the coagulation xe2x80x9ccascade.xe2x80x9d The cascade culminates in the conversion of soluble fibrinogen to insoluble fibrin which, together with platelets, forms a localized clot or thrombus which prevents extravasation of blood components. Wound healing can then occur followed by clot dissolution and restoration of blood vessel integrity and flow.
The events which occur between injury and clot formation are a carefully regulated and linked series of reactions. In brief, a number of plasma coagulation proteins in inactive proenzyme forms and cofactors circulate in the blood. Active enzyme complexes are assembled at an injury site and are sequentially activated to serine proteases, with each successive serine protease catalyzing the subsequent proenzyme to protease activation. This enzymatic cascade results in each step magnifying the effect of the succeeding step. For an overview of the coagulation cascade see the first chapter of xe2x80x9cThombosis and Hemorrhagexe2x80x9d, J. Loscalzo and A. Schafer, eds., Blackwell Scientific Publications, Oxford, England (1994).
While efficient clotting limits the loss of blood at an injury site, inappropriate formation of thrombi in veins or arteries is a common cause of disability and death. Abnormal clotting activity can result in and/or from pathologies or treatments such as myocardial infarction, unstable angina, atrial fibrillation, stroke, renal damage, percutaneous translumenal coronary angioplasty, disseminated intravascular coagulation, sepsis, pulmonary embolism and deep vein thrombosis. The formation of clots on foreign surfaces of artificial organs, shunts and prostheses such as artificial heart valves is also problematic.
Approved anticoagulant agents currently used in treatment of these pathologies and other thrombotic and embolic disorders include the sulfated heteropolysaccharides heparin and low molecular weight (LMW) heparin. These agents are administered parenterally and can cause rapid and complete inhibition of clotting by activation of the thrombin inhibitor, antithrombin III and inactivation of all of the clotting factors.
However, due to their potency, heparin and LMW heparin suffer drawbacks. Uncontrolled bleeding as a result of the simple stresses of motion and accompanying contacts with physical objects or at surgical sites is the major complication and is observed in 1 to 7% of patients receiving continuous infusion and in 8 to 14% of patients given intermittent bolus doses. To minimize this risk, samples are continuously drawn to enable ex vivo clotting times to be continuously monitored, which contributes substantially to the cost of therapy and the patient""s inconvenience.
Further, the therapeutic target range to achieve the desired level of efficacy without placing the patient at risk for bleeding is narrow. The therapeutic range is approximately 1 to less than 3 ug heparin/ml plasma which results in activated partial thromboplastin time (aPTT) assay times of about 35 to about 100 seconds. Increasing the heparin concentration to 3 ug/ml exceeds the target range and at concentrations greater than 4 ug/ml, clotting activity is not detectable. Thus, great care must be taken to keep the patient""s plasma concentrations within the therapeutic range.
Another approved anticoagulant with slower and longer lasting effect is warfarin, a coumarin derivative. Warfarin acts by competing with Vitamin K dependent post-translational modification of prothrombin and other Vitamin K-dependent clotting factors.
The general pattern of anticoagulant action,in which blood is rendered non-clottable at concentrations only slightly higher than the therapeutic range is seen for warfarin as well as for heparin and LMW heparin. Clearly, a need exists for an anticoagulant agent which is efficacious in controlling thrombotic and embolic disorders yet does not cause uncontrolled bleeding or its possibility.
Accordingly, one aspect of the present invention is a method for inhibiting thrombosis in an animal comprising administering an effective dose of an anti-coagulation factor monoclonal antibody having self-limiting neutralizing activity.
Another aspect of the invention is an anti-coagulation factor monoclonal antibody having self-limiting neutralizing activy against the coagulation factor.
Another aspect of the invention is a monoclonal antibody having the identifying characteristics of SB 249413, SB 249415, SB 249416, SB 249417, SB 257731, SB 257732, 9E4(2)F4 or 11G4(1)B9.
Another aspect of the invention is a hybridoma cell line having the identifying characteristics of 9E4(2)F4 or 11G4(1)B9.
Another aspect of the invention is a neutralizing Fab fragment or F(abxe2x80x2)2 fragment thereof, produced by deleting the Fc region of the monoclonal antibodies of the invention.
Another aspect of the invention is a neutralizing Fab fragment or F(abxe2x80x2)2 fragment thereof, produced by chain shuffling whereby the Fd heavy chain of the monoclonal antibodies of the invention is expressed in a murine light chain filamentous phage Fab display library.
Another aspect of the invention is a neutralizing Fab fragment or F(abxe2x80x2)2 fragment thereof, produced by chain shuffling whereby the light chain of the monoclonal antibodies of the invention is expressed in a murine heavy chain filamentous phage Fab display library.
Another aspect of the invention is an immunoglobulin heavy chain complementarity determining region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9 and 10.
Another aspect of the invention is an immunoglobulin light chain complementarity determining region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13 and 14.
Another aspect of the invention is an altered antibody comprising a heavy chain and a light chain, wherein the framework regions of said heavy and light chains are derived from at least one selected antibody and the amino acid sequences of the complementarity determining regions of each said chain are derived from an anti-coagulation factor monoclonal antibody having self-limiting neutralizing activity against the coagulation factor.
Another aspect of the invention is a chimeric antibody comprising a heavy chain and a light chain, said antibody characterized by inhibiting the function of intrinsic or common pathway coagulation factors in a self-limiting manner, wherein thrombosis is inhibited and limited modulation of coagulation is produced, wherein the constant regions of said heavy and light chains are derived from at least one selected antibody and the amino acid sequences of the variable regions of each said chain are derived from an anti-coagulation factor monoclonal antibody having self-limiting neutralizing activity against the coagulation factor.
Yet another aspect of the invention is a pharmaceutical composition comprising the humanized antibodies or chimeric antibody of the invention and a pharmaceutically acceptable carrier.